The present invention relates to a composition and method for enhancing paracellular transport across cell layers in an animal. More particularly, the present invention provides a composition and method comprising an antisense oligonucleotide hybridizable with a region of messenger RNA coding for the protein occludin (occludin mRNA) which, when hybridized to occludin mRNA, interferes with translation such that occludin function is disrupted and paracellular permeability is increased across an epithelial cell layer or an endothelial cell layer in an animal.
Transepithelial and transendothelial transport relate to the movement of solutes across a cell layer. In transcellular transport, solutes move both through and between cells. Movement of solutes through cells, for example across the lumenal and basolateral membranes of epithelial cells, requires transcellular transport. Transcellular transport may be active or passive depending on the solute in question. Active transcellular transport is carrier mediated and energy dependent, and permits solutes to move against their electrochemical gradients. Passive transcellular transport depends on electrochemical gradients generated by active transcellular transport and on the permeability of the cell membrane to the solute.
Movement of solutes between cells, through the tight junctions which bind cells together into a layer as with the epithelial cells of the gastrointestinal tract, is termed paracellular transport. Paracellular transport is passive. Paracellular transport depends on electrochemical gradients generated by transcellular transport and on solvent drag through tight junctions. Tight junctions form an intercellular barrier which separates the apical and basolateral fluid compartments of a cell layer. Movement of a solute through a tight junction from apical to basolateral compartments depends on the xe2x80x9ctightnessxe2x80x9d of the tight junction for that solute.
The xe2x80x9ctightnessxe2x80x9d of tight junctions varies among different epithelial cell layers. For example, in the gastrointestinal tract of the human, tight junctions in the colon are tighter than those in the ileum. In the kidney of the human, tight junctions in the ascending limb of the Loop of Henle are tighter than those in the proximal tubule. The xe2x80x9ctightnessxe2x80x9d of tight junctions also varies among different endothelial cell layers. For example, in the gastrointestinal tract, capillary endothelial cells have irregular tight junctions between adjacent cells enabling passage of some solutes between the cells. In the brain and spinal cord of the human, capillary endothelial cells have virtually continuous tight junctions between the adjacent cells, almost completely preventing passage of solutes between the cells.
Epithelial cell layers and endothelial cell layers present a significant barrier to the movement of solutes across cell layers. This can result in significant problems or barriers for drug absorption regardless of the route of administration of the drug. Therefore, new strategies for delivering drugs across epithelial and endothelial cell layers are needed.
Occludin is a xcx9c65 kD integral membrane protein which is localized at tight junctions in epithelial cell layers and in endothelial cell layers (Furuse et al., J. Cell Biol. 123:1777-1788, 1993). Occludin functions to seal the tight junctions formed between cells in a cell layer. Because occludin is required to maintain the integrity of tight junctions, modulation of occludin synthesis would be desirable to enhance the paracellular permeability of cell layers and, thereby, the movement of solutes, including drugs, across these cell layers.
Thus far, the nucleotide sequences of full length cDNA encoding occludin from human (SEQ ID NO:1), mouse (SEQ ID NO:2), dog (SEQ ID NO:3), chicken (SEQ ID NO:4) (Furuse et al., J. Cell Biol. 123:1777-1788, 1993) and kangaroo rat have been described (Ando-Akatsuka et al., J. Cell Biol. 133:43, 1996). The amino acid sequences of mammalian occludins from human, mouse and dog show approximately 90% homology, whereas the amino acid sequences from mammalian, chicken and rat kangaroo occludins show approximately 50% homology (Ando-Akatsuka et al., J.Cell Biol. 133:43, 1996).
The occludin protein comprises four transmembrane domains, a long carboxyl-terminal cytoplasmic domain, a short amino-terminal cytoplasmic domain, two extracellular loops and one intracellular turn. The nonpolar nature of the extracellular domains and the conservation of their sequences among human, mouse, dog, chicken and kangaroo rat occludin suggest the extracellular domains are important for occludin function.
In view of the location of occludin within tight junctions and the putative structure of occludin, two extracellular loops which face into the tight junction space, transient interference with either occludin synthesis or occludin function may result in a transient increase in paracellular permeability. Such a transient increase in paracellular permeability could enable an increase in drug absorption by paracellular transport across a cell layer with minimal toxic effects.
An antisense oligonucleotide is a sequence of single stranded DNA or RNA synthesized by chemical means in vitro which is xe2x80x9ccomplementaryxe2x80x9d in sequence to a specific intracellular target DNA or RNA. Antisense oligonucleotides offer the potential to block the expression of specific genes or translation of specific mRNAs within cells. The hydrogen bonding of an antisense oligonucleotide to its complementary mRNA may prevent or block the translation of the mRNA to yield the coded protein by steric hindrance. Alternatively, the interaction between an antisense oligonucleotide and its complementary mRNA within mammalian cells may induce the destruction of that mRNA by RNase H or by other unknown or uncharacterized RNases.
Therefore, what is needed is a composition and method for transiently interfering with occludin mRNA translation so that occludin synthesis and, thereby, occludin function are transiently disrupted and paracellular permeability is transiently enhanced.
The present invention satisfies the above need by providing a composition and method comprising an oligonucleotide hybridizable with a region of occludin mRNA, or of occludin DNA, which interferes with the translation of the mRNA, or the transcription of the DNA, when hybridized to the mRNA, or the DNA, such that the synthesis of occludin is down-regulated. More specifically, the composition and method of the present invention provides an antisense oligonucleotide which hybridizes with a region of occludin mRNA and, when hybridized to a region of occludin mRNA, interferes with its function such that occludin synthesis is disrupted. This antisense oligonucleotide, when administered in an effective concentration to an animal, including a human, transiently interferes with occludin translation so that occludin function in tight junctions is transiently disrupted, and so that the permeability of the cell layer to solutes, including drugs, is transiently increased.
Therefore, it is an object of the present invention to provide a composition and method for transiently enhancing paracellular transport across a cell layer in an animal including a human.
It is another object of the present invention to provide a composition and method for transiently disrupting the integrity of the tight junctions of an epithelial cell layer in an animal including humans.
It is another object of the present invention to provide a composition and method for decreasing the amount of occludin in the tight junctions of a cell layer.
It is another object of the present invention to provide a composition and method for disrupting the function of occludin in the tight junctions of a cell layer.
It is another object of the present invention to provide a composition and method for transiently disrupting the integrity of the tight junctions of an endothelial cell layer in an animal including a human.
It is another object of the present invention to provide a composition and method for enhancing drug movement across the gastrointestinal epithelium into the systemic circulation of an animal including a human.
It is another object of the present invention to provide a composition and method for enhancing drug movement from the systemic circulation into the interstitial space in an animal including a human.
It is another object of the present invention to provide a composition and method for enhancing drug movement across the pulmonary epithelium into the pulmonary circulation of an animal including a human.
It is another object of the present invention to provide a composition and method for enhancing drug uptake across the blood brain barrier in an animal including a human.
It is another object of the present invention to provide a composition and method for transiently interfering with the translation of occludin mRNA.
It is another object of the present invention to provide a composition and method for transiently interfering with the transcription of the occludin gene.
It is another object of the present invention to provide an antisense oligonucleotide composition that is stable.
It is another object of the present invention to provide an antisense oligonucleotide composition that is able to enter the target cells.
It is another object of the present invention to provide an antisense oligonucleotide composition that is retained by the target cells.
It is another object of the present invention to provide an antisense oligonucleotide composition that is able to interact with its cellular target.
It is another object of the present invention to provide an antisense oligonucleotide composition that interacts specifically with its cellular target.
It is another object of the present invention to provide an antisense oligonucleotide composition that demonstrates low toxicity.
It is another object of the present invention to invention to provide an antisense oligonucleotide composition that can be synthesized easily.
These and other object of the invention will become apparent from a review of the present specification.